Maintenance of arterial blood gas and acid-base homeostasis requires that a change in metabolism be matched by a proportional change in alveolar ventilation. Leptin, a cytokine hormone, has a central role in energy balance and has been implicated as an important contributor to the matching of ventilation to an aspect of metabolism in both mice and humans. This is clinically relevant as, for example, a subset of obese humans is resistant to leptin and hypoventilate with both an increase in arterial PCO2 and decrease in arterial PO2 (obesity hypoventilation syndrome, OHS). It is generally believed that leptin stimulates breathing through a CNS mechanism. However, multiple nuclei within the CNS contain leptin receptor expressing neurons (termed LepRb neurons) and there is almost no evidence as to which groups are involved in the stimulation of breathing. Based on preliminary studies and the literature we hypothesize that multiple specific groups of brainstem and hypothalamic LepRb neurons contribute to the respiratory stimulation. Additionally, peptide transmitters/modulators have been associated with many of these cell groups and pharmacologic manipulation of activity in these pathways could provide the basis for future development of clinically relevant pharmacological manipulation of activity in these pathways. Three Specific Aims will be addressed. In Aim 1, we will take advantage of Cre-loxP technology and optogenetic activation or silencing of specific LepRb neuronal groups in transgenic mice. Stimulation of breathing in response to selected activation of a specific LepRb neuronal group will suggest a role in breathing. Immunohistochemistry for the neuronal activity marker, c-Fos, will be used to identify cell groups that may participate as relay nuclei in pathways from specific LepRb neuronal groups to the CNS respiratory circuits. This potential role will be tested by using systemic leptin administration to stimulate breathing while determining whether bilateral inactivation of the target nucleus reduces the respiratory stimulation. In Aim 2, we will combine standard retrograde tracing with a novel transynaptic viral tracing method that crosses only 1 synapse to specify leptin-activated mono- and poly-synaptic pathways stimulating breathing. The peptide transmitters contained within these pathways will be identified immunohistochemically. In Aim 3, in vitro studies will define the impact of peptide transmitters within the leptin pathways on brainstem respiratory neurons and identify the cellular/molecular mechanisms underlying their influence. The combined studies will systematically identify CNS LepRb neuronal groups that stimulate breathing in response to systemic leptin administration. Additionally, paucisynaptic pathways mediating this influence will be revealed as will the identity of their peptide transmitters. Moreover, associated cellular/molecular mechanisms contributing to a stimulation of breathing will be defined. The findings could form the basis for the future development of pharmacotherapies for OHS patients.